Ion exchange cryptands covalently bound to substrates

ABSTRACT

One embodiment of the invention comprises an ion exchange composition formed by reacting unsaturated carbon to carbon moieties pendant from derivatized ion binding cryptands with a support substrate under free radical activation conditions to form a covalent bond therebetween.  
     In another embodiment, a cryptand ion exchange composition is made by covalently bonding unsaturated carbon to carbon moieties pendant from a derivatized ion binding cryptands with unsaturated carbon to carbon moieties pendant from a support substrate under free radical activation conditions to form covalent bond.

FIELD OF THE INVENTION

[0001] The present invention relates to cryptands covalently bound to asupport substrate for uses such as in ion exchange chromatography.

BACKGROUND OF THE INVENTION

[0002] Cryptands and other macrocyclic compounds such as crown ethers,spherands, cryptahemispherands, cavitands, calixarenes, resorcinorenes,cyclodextrines, porphyrines and others are well known. (ComprehensiveSupramolecular Chemistry Vol. 1-10, Jean-Marie Lehn—Chairman of theEditorial Board, 1996 Elsevier Science Ltd.) Many of them are capable offorming stable complexes with ionic organic and inorganic molecules.Those properties make macrocyclic compounds candidates for variousfields, for instance, catalysis, separations, sensors development andothers. Cryptands (bicyclic macrocycles) have extremely high affinity tometal ions. The cryptand metal ion complexes are more stable than thoseformed by monocyclic ligands such as crown ethers (Izatt, R. M., et al.,Chemical Reviews 91:1721-2085 (1991)). This high affinity of thecryptands to alkaline and alkaline earth metal ions in water makes themsuperior complexing agents for the processes where strong, fast andreversible metal ion binding is required. Examples of these processesinclude separation, preconcentration and detection of metal ions,analysis of radioactive isotopes, ion-exchange chromatography,phase-transfer catalysis, activation of anionic species and others.

[0003] Adding moieties with functionality to macrocyclic compoundspermits binding of the derivatized macrocycles onto support substratesto provide surface functionalization. Physical adsorption and covalentattachment are two common methods of binding. Cryptand adsorbed polymershave been reported as stationary phases for ion exchange chromatography(Lamb, J. D., et al., J. Chromatogr., 482:367-380 (1989); Niederhauser,T. L., et al., Journal of Chromatography A, 804:69-77 (1998); Lamb, J.D., et al., Talanta, 39(8):923-930 (1992); and Smith, R. G., et al.,Journal of Chromatography A, 671:89-94 (1994).

[0004] The majority of adsorbed materials have limited number ofapplications due to their incompatibility with the solvents that elutethe adsorbed functional layer. There is also a restriction on usingthese materials at elevated temperatures. Covalent attachment reducesthese problems. Previously reported substrates with covalently attachedmacrocycles include silica gel, polymeric resins, thin films and others(Blasius, E., et al., Pure & App. Chem. 54(11):2115-2128 (1982);Montanari, F., et al., British Polymer Journal, 16:212-218 (1984); U.S.Pat. No. 5,393,892 to Krakowiak, et al.; U.S. Pat. No. 4,943,375 toBradshaw, et al.; U.S. Pat. No. 5,968,363 to Riviello, et al.; JP PatentNo. 55018434A2 to Kakiuchi, et al.; JP Patent No. 59145022A2 to Fujine,et al.; JP Patent No. 61033220A2 to Fujine, et al.; JP Patent No.4346064A2 to Watanabe, et al.; and PCT Publication W099/28355 toDarling, et al.

[0005] Many strategies for the synthesis of macrocyclic compounds havebeen developed over the years (Comprehensive Supramolecular ChemistryVol. 1-10, Jean-Marie Lehn—Chairman of the Editorial Board, 1996Elsevier Science Ltd.; Krakowiak, K. E., et al., Israel Journal ofChemistry 32:3-13 (1992); Bradshaw, J S., et al., “Aza-CrownMacrocycles,” The Chemistry of Heterocyclic Compounds, Vol. 51, ed.Taylor, E. C., Wiley, New York, 1993; Haoyun, A., et al., ChemicalReviews 92:543-572 (1992)). However, the synthesis of functionalizedmacrocycles is difficult. Hydroxy, amino and carboxylic groups added tolinear precursors before the ring closure step are commonly usedfunctionalities for derivatization of macrocycles. Most of the syntheticprocedures imply protection of these groups prior to cyclization.Protected groups are chemically transformed into desired functions afterthe macroring is constructed (Krespan, C. G., Journal of OrganicChemistry 45:1177-1180 (1980); Montanari, F., et al., Journal of OrganicChemistry 47:1298-1302 (1982); Haoyun, A., et al., Journal of OrganicChemistry 57:4998-5005 (1992)). This methodology can impose considerablelimitations on synthesis and purification of functionalized macrocycles,especially bicyclic and polycyclic compounds. Synthetic difficulties canlead to low overall yields and high production costs of these materials.

[0006] Macrocyclic compounds containing allylic functionalities areknown from prior art (Krakowiak, K. E., et al., Journal of HeterocyclicChemistry 27:1011-1014 (1990)). Some of them were further hydrosilatedand attached to silica solid supports (Bradshaw, J. S., et al., Pure &Appl. Chem. 61:1619-1624 (1989); Bradshaw, J. S., et al., Journal ofInclusion Phenomena and Molecular Recognition in Chemistry 7:127-136(1989)). The synthesis of allyl containing [2.2.2] cryptand 1 has beenreported (Babb, D. A., et al., Journal of Heterocyclic Chemistry23:609-613 (1986)).

[0007] The methods for covalent attachment of the cryptands to polymericsubstrates are based mostly on the interaction of active layer of asubstrate, for example, benzyl chloride groups with hydroxyl or aminofunctionalized cryptand molecules (Montanari, F., et al., J. Org. Chem.,47:1298-1302 (1982); Montanari, F., et al., British Polymer Journal,16:212-218 (1984) and Montanari, F., et al., Tetrahedron Letters, No 52,5055-5058 (1979)). This interaction also involves the sideprocess—formation of the quaternary centers from the tertiary nitrogensof the macrocycle (Montanari, F., et al., British Polymer Journal,16:212-218 (1984). Quaternisation causes extended decomposition of themacrocycle via Hofmann degradation reducing the capacity of the anionexchange stationary phase. An amide group is another linker reported fora covalent functionalization of the substrates with cryptand molecules(Montanari, F., et al., British Polymer Journal, 16:212-218 (1984).Amides do not withstand the extremely high pHs used in anion exchangechromatography. Moreover, most of the described synthetic for producinghydroxyl or amino functionalized cryptands, are not practical to satisfythe requirements of industrial scale production.

[0008] There is a need to provide an improved method for covalentbonding of cryptands to a substrate for uses such as a chromatographicseparation medium to separate anions.

SUMMARY OF THE INVENTION

[0009] One embodiment of the invention comprises an ion exchangecomposition formed by reacting unsaturated carbon to carbon moietiespendant from derivatized ion binding cryptands with a support substrateunder free radical activation conditions to form a covalent bondtherebetween.

[0010] In another embodiment, a cryptand ion exchange composition ismade by covalently bonding unsaturated carbon to carbon moieties pendantfrom a derivatized ion binding cryptands with unsaturated carbon tocarbon moieties pendant from a support substrate under free radicalactivation conditions to form covalent bond.

BRIEF DESCRIPTION OF THE DRAWINGS

[0011]FIG. 1 is a schematic representation of an ion exchangecomposition according to the present invention.

[0012]FIGS. 2 and 3 are chromatograms illustrating uses of the ionexchange composition of the present invention.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0013] In one aspect of the present invention, an ion exchangecomposition is formed by reacting unsaturated carbon to carbon moietiespendant from a derivatized ion binding cryptand C with a supportsubstrate under free radical activation conditions.

[0014] In one embodiment, the cryptand pendant unsaturated carbon tocarbon moiety is on a styrenic group which has been appended to thecryptand by techniques such as described below. The properties ofcryptands are well known as ion exchange compositions. As set forth inthe publications described above, cryptands bind with cations such aspotassium, sodium, rubidium, calcium, strontium, barium, thallium and toa lesser degree with cesium, magnesium and lithium and form an anionexchanger according to well known principles. The concentration ofcomplexed cation is directly proportional to the binding constant K,also known as the stability constant. If these macrocycles are attachedto a support such as a conventional chromatographic stationary phase,and subsequently complexed with certain cations, anion exchangechromatography can be achieved.

[0015] One advantage of the reaction schemes of the present invention isthat individual multiple (e.g., 4×10¹⁴ to 4×10¹⁶ or more) strands ofderivatized macrocycle compounds (e.g., cryptands) per square meter canbe bound to each support particle and projecting therefrom asillustrated in FIG. 1. This provides a substantial quantity ofmacrocycle compounds in a format which is readily accessible to theapproach of both cationic and anionic species in an aqueous environment.Prior art based on adsorbed macrocycle compounds provides very limitedcapacity due to the restriction that this species must be adsorbed as amonolayer on a hydrophobic surface. Presenting the macrocycle compoundsas a brush polymer extending into the aqueous solution surrounding eachsupport particle, permits significantly better diffusion kinetics thancan be observed with adsorbed monolayer coatings of macrocyclecompounds, while at the same time allowing for significantly highercapacity than is possible with an adsorbed monolayer coating.Furthermore, positioning the macrocycle compounds in the proximity of ahydrophobic surface compromises the chromatographic performance ofmaterials based on adsorbed macrocycle compounds. Anionic hydrophobiccompounds exhibit poor chromatographic efficiency when that retentionsite is located in the proximity of a hydrophobic surface unless organicsolvent is added to the mobile phase. Of course, incorporating anorganic solvent into mobile phase flowing past the support particles isincompatible with the prior art adsorbed monolayer coatings because suchorganic solvent will slowly wash the adsorbed coating away. While thecomposite material derived from support particles which had beencovalently derivatized with macrocycle brush polymers are compatiblewith organic solvents, the high cost of organic solvents and the highcost of disposal of organic solvents make the use of organic solventshighly undesirable. Thus, the brush polymer configuration describedabove provides superior chromatographic properties without the need forthe addition of organic solvent and the incumbent disadvantages of itsuse.

[0016] In one preferred embodiment, the covalent bond is formed betweenthe cryptand pendant moieties and an unsaturated carbon to carbon moietypendant from the polymeric substrate under free radical conditions. Forexample, the cryptands can be bound to resin beads such as cross-linkedpolystyrene resin for use in a packed bed for an ion exchange column.The form of the bed and the characteristic composition of the resin canbe of the type sold by Dionex under the IONPAC® product line. In aparticularly effective ion exchange composition of this type, thecryptand is derivatized to include a pendant styrenic group which formsthe covalent bond.

[0017] In a further embodiment, the only ion binding moiety bound to thepolymeric substrate is a cryptand.

[0018] The vinyl moiety of the styrenic terminal group is readilyattached under free radical conditions to polymeric substrate particlesincluding terminal groups with ethylenic unsaturation as described inU.S. Pat. No. 5,865,994.

[0019] In another embodiment, the support substrate includes hydrogenatoms which are abstractable under hydrogen abstraction conditions toform a covalent bond with the cryptand pendant unsaturated moiety.Suitable conditions for this reaction include free radical activation asdisclosed in U.S. Pat. Nos. 6,074,541 and 5,792,331.

[0020] An anion exchange column of the foregoing type can perform avariety of anion exchange separations ranging from common anions such asfluoride, chloride, sulfate, nitrite, bromide, phosphate and nitrate;hydrophobic anions such as thiosulfate, iodide, thiocyanate andperchlorate; organic acids such as formic, acetic, glycolic, quinicacids. Polyvalent ions such as polyphosphates, oligonucleotides, andoligosacarrides at high pH can also be separated with this column.

[0021] The conditions for separation of anions using cryptands are wellknown. (Lamb, J. D., et al., J. Chromatogr., 482:367-380 (1989);Niederhauser, T. L., et al., Journal of Chromatography A, 804:69-77(1998); Lamb, J. D., et al., Talanta, 39(8):923-930 (1992); and Smith,R. G., et al., Journal of Chromatography A, 671:89-94 (1994). An exampleis shown in FIG. 2 where the separation of seven common anions isaccomplished using conventional suppressed anion exchange techniques.Here we see the separation of fluoride (peak 1), acetate/formate (peak2), chloride (peak 3), sulfate (peak 4), nitrite (peak 5), bromide (peak6), phosphate (peak 7), and nitrate (peak 8) using a mobile phase of 70millimolar sodium hydroxide.

[0022] As set forth above, in one embodiment a monomeric cryptand isgrafted onto the support substrate (stationary phase) using a freeradical initiator to produce a covalently bound functionality. Thisstationary phase is stable when used with aqueous or organic mobilephases. Bicyclic and tricyclic aliphatic and aromatic cryptand moleculescontaining nitrogen and oxygen heteroatoms or only oxygen heteroatoms oronly nitrogen heteroatoms or incorporated heterocyclic fragments formetal ion binding can also be applied as modifiers. Other polymerizablecryptand compounds such as their styrenic, allylic, acrylic, methacrylicand any other alkene and alkyne derivatives can be used for grafting.Once the cryptand is attached by a covalent bond by this or any of themethods possible to form such bonds, the cryptand can now be utilized asa stable functional group for anion exchange chromatography.

[0023] Anion exchange mobile phases are typically basic solutions suchas sodium or potassium hydroxide (or carbonate). Elution of anionsoccurs via competition between the mobile phase anion such as hydroxideor carbonate and the sample anions for cation exchange sites on thestationary phase. In conventional suppressed anion exchangechromatography, the associated cation typically plays a negligible rolein the process. In cryptand based anion exchange, the cation plays acrucial role. It generates the anion exchange site, and depending on thecation, determines the capacity and selectivity of the stationary phase.

[0024] For example, if a basic mobile phase such as potassium hydroxideis used with [2.2.2] cryptand resin, a high capacity stationary phase iscreated since potassium has a high binding constant relative to sodiumor lithium. If a mobile phase such as sodium hydroxide is used, a lowercapacity stationary phase is now created since sodium has a lowerbinding constant. A low capacity phase is generated if lithium hydroxideis used as the mobile phase since lithium has a low affinity for the[2.2.2] cryptand. The capacities decrease as expected based on thebinding constants of the 2,2,2 cryptand with potassium, sodium, andlithium cations.

[0025] According to the present invention, the macrocycle is permanentlyattached to a support substrate and will not slowly bleed off an ionexchange column when using aqueous eluents or eluents which containsolvents.

[0026] Another advantage is the ability to rapidly restore columns whoseperformance has been compromised by contamination with polyvalent ions.This can be done by using a very low capacity mode with no substantialaffinity for these ions. Under these conditions, the polyvalentcontaminants easily elute off the column and the original performance isrestored.

[0027] Conventional anion exchange columns have a fixed capacity andselectivity, which cannot be adjusted. In the present invention, one hasthe ability to vary the anion exchange capacity by changing the cationcomplexed with the cryptand. Selectivity to a lesser degree can also beadjusted by these mobile phase changes. This feature allows the user tomodify the column performance relative to the sample being analyzedsimply by the choice of mobile phase. Not only can the capacity beadjusted for a single analysis, but it can also be varied during ananalysis by using a higher capacity mobile phase at the beginning andthen changing to a lower capacity format by either a step change or agradient. An example of this is shown in FIG. 3 where the initial mobilephase is sodium hydroxide and then a step change is made to a lithiumhydroxide mobile phase, which is a very low capacity form. The firstpeaks eluting off are low valency polyphosphates and the run ends withthe elution of high valency ions.

[0028] The benefit of this approach is that it allows one to elute veryhigh valency ions without having to increase the mobile phase ionicconcentration. The total ionic concentration throughout the run is only30 mM, a relatively low concentration. This technique is known as“gradient capacity ion chromatography” (2,3) chromatography (Lamb, J.D., et al., J. Chromatogr., 482:367-380 (1989); Lamb, J. D., et al.,Talanta, 39(8):923-930 (1992);

[0029] Traditional anion exchange columns require very highconcentration mobile phases when analyzing polyvalent ions such aspolyphosphates, oligosacarrides, oligonucleotides, etc. Theconcentrations needed sometimes exceed the ability of a suppressor tosuppress. By using a cryptand based column one can eliminate largebaseline changes often seen with steep gradients or step changes withconventional approaches.

[0030] One mode for forming a derivatized cryptand is disclosed inBordunov, et al. application, entitled “A Derivatized MacrocycleCompound for Covalent Bonding to a Substrate and Method of Forming andUse,” filed simultaneously herewith. As disclosed therein, cryptands Care derivatized by the use of a sulfur-containing derivatizing agent toform a product which includes a terminal functional moiety R bound,directly or indirectly, to C. R is capable of covalently binding to asupport substrate or of being converted into a form capable ofcovalently binding to such a substrate. In general, the derivatized Chas the following structure:

[0031] wherein C is a molecular framework monocyclic or polycycliccryptand moiety containing at least 12 atoms in each cycle;

[0032] L is a substituted or unsubstituted carbon chain linkercovalently bound to C including at least one carbon atom in a structureselected from the group consisting of an aliphatic, aromatic orheterocyclic linker including heteroatoms substituted for hydrogen atomson the linker;

[0033] R is a terminal functional moiety capable of covalent binding toa support substrate or of being converted into a functional moietycapable of covalent binding to a solid support substrate;

[0034] X and Y are moieties selected from the group consisting ofprotons, aliphatic groups, aromatic groups, optionally includingheteroatoms, substituted for hydrogen atoms in the moieties, selectedfrom the group consisting of oxygen, nitrogen, sulfur, or phosphorusheteroatoms; and S is sulfur.

[0035] Suitable macrocyclic compounds C are monocyclic, bicyclic,tricyclic or polycyclic molecular frameworks. Examples of suchmacrocyclic compounds include crown ethers, cryptands, spherands,cryptahemispherands, cavitands, calixerenes, resorcinorenes,cyclodextrines and porphyrines such as of the type described above.According to the invention, the R group in structure (1) can becovalently bound in one or more steps to a support substrate Z to formthe following structure:

[0036] the transformation of R or functional group derived from Rresults in the formation of linker R₁ during functionalization of Z. R₁is a covalent linker between S and Z. It can be a substituted orunsubstituted carbon chain including at least one carbon atom in astructure selected from the group consisting of an aliphatic, aromaticor heterocyclic linker including heteroatoms substituted for hydrogenatoms on the linker. Although R₁ is illustrated to be directly bound toZ, it can be bound to an intermediate compound which is capable ofcovalent binding to Z, as shown in reactant scheme (5). Thus, the -R₁-Zlinkage of structure (3) encompasses a direct and indirect bonding anddoes not exclude such an intermediate linkage.

[0037] Any support substrate Z can be used so long as C in structures(1)-(3) is capable of performing its desired function, e.g., to serve asan ion exchanger. One form of structure (3) is a packed bed of particlesof derivatized macrocycle compound covalently bound to substrate Z.Suitable substrates include organic or inorganic materials such ascross-linked and uncross-linked polymers, resins, organic or inorganicmonoliths, sol-gels, other forms of gels such as silica gels, inorganicsupports such as zeolites, aluminum oxide, titanium dioxide, zirconiumbased supports, glasses, carbon black, activate carbon, carbonnanotubes, fibers, pyrolized materials, organic and inorganic crystals,liquid crystals, colloids, nanoparticles, organic and inorganic gels,latexes, foams, membranes and films. Also, Z may be in the form ofmonolayers such as surfaces of chips, silicon wafers, the walls ofcapillaries used for gas, liquid, capillary and ion exchangechromatography, capillary electrophoresis, separation, extraction, solidphase extraction, filtration, purification, transport, complexation,molecular and ion recognition, concentration, sensing an analysis oforganic and inorganic molecules and ions and also for catalysis, phasetransfer catalysis, solid phase synthesis or for other applications.

[0038] One particularly useful macrocycle comprises a cryptand bound toa support substrate such as resin copolymer particles in a flow-throughion exchange bed, e.g., using the cryptand functional bed for anionexchange chromatography.

[0039] According to one embodiment of the present invention, amacrocyclic compound C is derivatized to include a pendant reactivemoiety such as an allylic group by well known methods as describedabove. C is defined to include such reactive moieties which are capableof bonding to HSR as described below. Thus, the HSR reagent iscovalently bound to C to form an intermediate product of the type shownin structure (1) in which the R group is covalently bound to themacrocycle indirectly through the sulfur atom S. As set forth above, theR group can be in a functional form suitable for direct or indirectcovalent attachment to the support substrate in a single or multiplesteps. Scheme (4) illustrates the derivatization of macrocyclic compound(2) with HSR under the conditions of free radical addition. In aparticular case compound (2) is the cryptand with the allylic pendantmoiety.

[0040] In structure (1), S is connected to the macrocycle C through theintermediate linker L including at least one carbon atom. In structure(2), L is disposed between C and an unsaturated carbon to carbon bond Uwhich interconnects L and the terminal carbon atom bound to Y. Thepurpose of linker L is to incorporate function U into the macrocycle C.

[0041] In one embodiment, the unsaturated carbon to carbon bond, e.g., a—C═C (double bond) or —C/C— ( triple bond) described as U in (2) servesas the reacting site for free radical addition of HSR to C through theterminal group U distal to C. The unsaturation is preferably provided bythe double bond, e.g., a terminal allyl group. Linker L may be attachedto C at any site that does not significantly affect the ability of themacrocycle to provide the desired function, e.g., to complex with an ionof interest. Thus, for a cryptand, the attachment would notsignificantly affect binding of the cation or its associated anion. Asdescribed above, the backbone of the linker L is preferably from about 1to about 20 atoms in length, preferably from 3 to 8 atoms in length. Thelinker chain may be straight chained or branched and it may also includesaturated or unsaturated carbon atoms for heteroatoms substituted forhydrogen atoms on the linker including oxygen, nitrogen, sulfur orphosphorus. Usually the linker group will contain from 1 to 3heteroatoms. The heteroatoms may be placed in the linker chain atpositions where they will have no significant adverse affect on the ionseparation characteristics of the composition. The linker group L can besimilar to the corresponding linker L in U.S. Pat. No. 5,865,994,incorporated herein by reference.

[0042] Conditions suitable for free radical attachment of the HSR groupto a pendant unsaturated group on the macrocycle by free radicalinitiation are well known in the art. For example see Griesbaum, K,Angew. Chem. Internat. Edit. Vol.9, No.4, 273-287 (1970).

[0043] In one embodiment of the reaction scheme (4), R is in a formcapable of direct covalent attachment to a support substrate withoutconverting R to a form capable of covalent attachment. The conversion ofR such as protection/deprotection reactions might be necessary to keep Rintact during the reaction (4). Another reason for an optionalprotection/deprotection of R is to prevent the interference of group Rwith the course of reaction (4). The example of theprotection/deprotection of R is using a carboxylic acid in a form ofester protected R group in reaction (4) followed by its conversion (deprotection) to carboxylic acid upon hydrolysis, prior to itsattachment to a substrate. The groups R suitable for a direct covalentattachments to a support substrate with the possible use ofprotection/deprotection include proton, amines, epoxides, aldehydes,ketones, alcohols, phenols, thiols, carboxylic acids, thiocarboxylicacids, amides and esters of carboxylic and thiocarboylic acids,phosphoric and phosphoric acids, esters of sulfonic acids.

[0044] Reaction schemes such as reported earlier (Montanari, F., et al.,J. Org Chem., 47:1298-1302 (1982); Montanari, F., et al., BritishPolymer Journal, 16:212-218 (1984) and Montanari, F., et al.,Tetrahedron Letters, No 52, 5055-5058 (1979)) may be used for a directfunctionalization of a support substrate with the cryptand modifier. Oneof the described approaches is the reacting hydroxymethyl functionalizedcryptand with the chrolomethyl polystyrene polymer in presence of thebase. Some disadvantages of this and other previously reported methodsfor a direct covalent attachment of the cryptands are described above.

[0045] There are at least two ways of indirect attachment of (1) to asupport substrate. The first method is the conversion of R to a groupcapable of covalent binding to a substrate under non radical conditionsto form structure (3). The example of this approach can be a conversionof (1) where R is the alcohol moiety. The alcohol group can be easilytransformed into a tosyl or mesyl derivative which reacts with thedeprotonated hydroxyl groups of a support substrate to form a covalentlink R₁. Functional groups that can be prepared by conversion of R forthe further non radical covalent attachment to a substrate includeamines, epoxides, aldehydes, ketones, alcohols, phenols, thiols,carboxylic acids, thiocarboxylic acids, amides and esters of carboxylicand thiocarboxylic acids, phosphoric and phosphoric acids, esters ofsulfonic acids, acyl halides, alkyl and aryl halides and activatedcarboxylic acids.

[0046] A second method of indirect attachment of (1) to a supportsubstrate is the conversion of group R into polymerizable moietyfollowed by its covalent binding to a substrate under free radicalconditions. The indirect attachment of the below specific reactionscheme (5) illustrates a two-step procedure in which —SR in the HSRreagent first is bonded through linker L to C. Then, in a second step,the bound R is reacted with another reagent to form a pendant group on Rcapable of covalent binding to a substrate via radical process. In thefollowing specific reaction scheme (5), the pendant group is anethylenically unsaturated (vinyl) group which can be bound to thesupport substrate under free radical activation conditions.

[0047] Scheme (5). Synthesis of the amino and styryl [2.2.2] cryptandsvia radical addition of 2-aminoethanethiol hydrochloride to allylderivatized [2.2.2] cryptand.

[0048] Referring specifically to reaction scheme (5), an allylderivative of [2.2.2] cryptand 1 is first formed by known chemistry asdescribed above. Then, it is covalently bound to the HSR reagent(2-aminoethanethiol hydrochloride) through the allyl group to form aterminal amino group R, such as by free radical conditions such as theexposure to UV or other irradiation and/or by addition of peroxides, azocompounds, etc., e.g., as illustrated in the review of Griesbaum, K.,Angew. Chem. Internat. Edit, Vol.9, No.4, 273-287 (1970). Thereafter,amino group R is converted to a function capable of binding to asubstrate under free radical conditions e.g., a vinyl group. Asillustrated in the reaction scheme (5), the first step of thisconversion is the interaction of amino cryptand 2 with4-vinylbenzaldehyde. A Schiff base is the intermediate product of thisreaction (not shown on the scheme). On the second step, Schiff base isreacted in situ with NaBH₄ to give [2.2.2] cryptand 3 functionalizedwith polymerizable styryl moiety. This approach allows ready conversionof macrocycles into functionalized molecules for their further covalentattachment or incorporation into or onto various substrates using freeradical process such as grafting and coating.

[0049] In the foregoing description, C is functionalized by free radicaladdition of thiols (schemes (4) and(5)). Advantages of this processinclude the following:

[0050] 1. Synthesis of allyl cryptand 1 described in prior art is thesuperior method to build the macrocyclic framework of [2.2.2] 1 cryptandhaving pendant function for further attachment. The use of allyl groupalleviates the need for protection/deprotection steps during thesynthesis of the cryptands. Most of the prior art examples of cryptandfunctionalization are based on protected intermediates with longerroutes for their synthesis effecting the total outcome of the process .The method developed on a base of the allyl precursor allows 100-200 gscale production of the functionalized [2.2.2] cryptand. This isunusually large amount for all described methods of the cryptandsynthesis.

[0051] 2. The methods for a conversion of allyl cryptand to morereactive functional molecules are limited. For instance, the authors whofirst synthesized [2.2.2] allylic cryptand failed to convert the allylgroup to hydroxy group. The thiol addition was found very effective forchemical transformation of relatively inert allyl group to reactiveamine. The amino group itself is highly efficient for thefunctionalization of the substrates, however the requirements for theanion exchange stationary phases are in favor of the materialsfunctionalized under conditions of radical polymerization.

[0052] 3. Allylic group has lower reaction ability compared to styrenicfragment under conditions of radical polymerization. Thus, allylicmonomers very often do not provide the required grafting efficiency andlead to low capacity stationary phases. The developed thiol additionallowed efficient transformation of the allyl group to styrenic moietyvia two step process (5). Allylic cryptand 1 converted to a styrenicderivative 3 now can be efficiently grafted from the surface of thesupport providing novel high capacity anion exchange stationary phase.

[0053] 4. Chemical stability of the stationary phases used in ionexchange chromatography is of great importance. The extreme pHs at whichthe ion exchange chromatography is performed impose considerablelimitations on chemistry of the functional monomers and linkersconnecting the ion exchange sites with the stationary phase. Thedeveloped thiol addition method followed by grafting polymerizationprovides extremely stable anion exchange materials on a base of thecryptand functionalized resins. These stationary phases can be operatedat pH 1-14 at elevated temperatures. The ruggedness and reproducibilityof these phases after subjecting them to such harsh conditions aresuperior to similar characteristics of the existing anion exchangematerials.

[0054] In the reaction scheme (5), the illustrated R group is NH₂ whichreacts to form styrenic [2.2.2] cryptand 3 in which the pendant vinylgroup can form a covalent bond with a corresponding vinyl group on acopolymer resin support substrate under free radical conditions asillustrated in Example 3 and FIG. 1.

[0055] One advantage of the foregoing reaction schemes is thatindividual multiple (e.g., about 4×10¹⁴ to 4×10¹⁶ or more) strands ofderivatized macrocycle compounds (e.g., cryptands) per square meter canbe bound to each support particle and projecting therefrom asillustrated in FIG. 1. This provides a substantial quantity ofmacrocycle compounds in a format which is readily accessible to theapproach of both cationic and anionic species in an aqueous environment.Prior art based on adsorbed macrocycle compounds provides very limitedcapacity due to the restriction that this species must be adsorbed as amonolayer on a hydrophobic surface. Presenting the macrocycle compoundsas a brush polymer extending into the aqueous solution surrounding eachsupport particle, permits significantly better diffusion kinetics thancan be observed with adsorbed monolayer coatings of macrocycle compoundswhile at the same time allowing for significantly higher capacity thanis possible with an adsorbed monolayer coating. Furthermore, positioningthe macrocycle compounds in the proximity of a hydrophobic surfacecompromises the chromatographic performance of materials based onadsorbed macrocycle compounds. Anionic hydrophobic compounds exhibitpoor chromatographic efficiency when that retention site is located inthe proximity of a hydrophobic surface unless organic solvent is addedto the mobile phase. Of course, incorporating an organic solvent intomobile phase flowing past the support particles is incompatible with theprior art adsorbed monolayer coatings because such organic solvent willslowly wash the adsorbed coating away. While the composite materialderived from support particles which had been covalently derivatizedwith macrocycle brush polymers are compatible with organic solvents, thehigh cost of organic solvents and the high cost of disposal of organicsolvents make the use of organic solvents highly undesirable. Thus, thebrush polymer configuration described above provides superiorchromatographic properties without the need for the addition of organicsolvent and the incumbent disadvantages of its use.

[0056] Adsorbed macrocycle monolayer coatings of the prior art have beenshown to be useful with macrocycle ion binding constants as low as 60.However, surprisingly similar macrocycles when constructed in the formof a covalently attached brush polymer coating failed to exhibit anyuseful ion binding characteristics in 100% aqueous environments.Apparently, applying a macrocycle as an adsorbed monolayer coating on ahydrophobic surface exposes the macrocycle to a substantially lowerdielectric environment than the aqueous environment above the surface.As such, ion binding affinities of adsorbed monolayer coatings aresubstantially higher than the equivalent macrocycle in a 100% aqueousenvironment. Therefore, in order to produce a useful material in thehighly desirable covalently bound brush polymer format, macrocycles musthave higher ion binding constants in order to provide useful ion bindingcharacteristics in 100% aqueous environments.

[0057] The vinyl moiety of the styrenic terminal group is readilyattached under free radical conditions to polymeric substrate particlesincluding terminal groups with ethylenic unsaturation as described inU.S. Pat. No. 5,865,994 to form structure (3).

[0058] In the specific two-step reaction (scheme 5) discussed above forbonding R to the macrocycle, R is illustrated in the form of an amineconverted to an unsaturated carbon-carbon bond, specifically a vinylgroup. Other terminal functional groups may be employed so long as theycan be covalently bonded to a desired support substrate.

[0059] According to the invention, one useful effective composition is aderivatized macrocycle compound with sufficient ion exchange propertiesto separate charged molecules. A particularly effective derivatizedmacrocycle compound of this type is a cryptand bound to a supportsubstrate with anion exchange properties. By anion exchange propertiesis meant the capability of performing anion exchange chromatography.Cryptands are particularly effective for anion separation because theyprovide ion binding characteristics sufficient for binding alkaline andalkaline earth metals under alkaline conditions and yet readily releasethese ions under acidic conditions, providing a convenient means ofconverting the cryptand from one alkali metal form to another.Comprehensive Supramolecular Chemistry Vol. 1-10, Jean-MarieLehn—Chairman of the Editorial Board, 1996 Elsevier Science Ltd; Izatt,R. M., et al., Chemical Reviews 91:1721-2085 (1991)). Significantlyhigher affinity of the cryptands to alkaline metal ions as compared tothe same property of the crown ethers for instance, provides better ionretention characteristics to the anion exchange stationary phasesfunctionalized with the cryptand modifiers. In fact, due to lowcapacities of the crown ether based materials they are impractical foruse as the anion exchange chromatographic phases with 100% aqueouseluents. Most of the industrial applications for ion exchangechromatographic processes are developed with the aqueous eluents,therefore the cryptand functionalized materials are superior to otherknown metal ion complexing agents.

[0060] The anion exchange capacity of the above cryptand-grafted resincan range from about 15 to 2000 microequivalents per gram with apreferable range from about 100 to 300, more preferably from about 120to 225 and yet more preferably from about 150 to 200 microequivalentsper gram.

[0061] One suitable support particle is a macroporous polymeric resin, eg, vinylbenzene ethylene, cross-linked with divinylbenzene. Suitablemacroporous resins are illustrated in U.S. Pat. No. 4,224,415.

[0062] In another embodiment of the invention, a hydrophilic layer maybe attached to Z which forms the covalent attachment with thederivatized macrocycle. This has the advantage of reducing hydrophobicinteraction between hydrophobic analytes and the surface to which themacrocycles are covalently attached. This reduces the need for additionof solvent to the mobile phase which as noted above is undesirable.Suitable procedures are set forth in Examples 3 and 5.

[0063] The foregoing description illustrates functionalizing thecryptand to include unsaturation for covalent binding to Z by a thiolreaction. However, such functionalizing can also be accomplished byother. Other elements besides sulfur can be attached to unsaturatedcarbon-carbon bonds under proper conditions of radical initiation. Theseelements are silicon and germanium atoms having at least one hydrogenatom bound to them directly. Substituted carbons, for example,halogenated carbons can also be added to unsaturated carbon-carbon bondsvia radical process. Elements mentioned above can be a part of themolecular structure (1) where sulfur is substituted for one of theseelements bound directly or indirectly to R.

[0064] All patents and publications referred to herein are incorporatedby reference.

[0065] Further details of the invention are illustrated in the followingnon-limiting examples.

EXAMPLE 1

[0066] This example describes a two-stage synthesis of a derivatizedcryptand according to reaction scheme (5).

Methods for Derivatization of Allyl Cryptand 1 Procedure for theSynthesis of Cryptands 2 and 3 Scheme (5)

[0067] The procedure for the synthesis of allylic [2.2.2] cryptand isbased on the reported method (Babb, D. A., et al., Journal ofHeterocyclic Chemistry 23:609-613 (1986)). 12 g of allyl-derivatizedcryptand was dissolved in 70 ml of ethanol and 12 g of2-aminoethanethiol hydrochloride was added to the reaction mixture.Reactor was purged with nitrogen. Solution was brought to reflux and 65mg of AIBN was added. UV irradiation at 254 nm wave length was applied.Reaction mixture was stirred under reflux and irradiated for eighthours. After every two hour period, a new portion of AIBN (65 mg) wasadded to the reaction mixture. Reaction is being monitored using TLC onneutral aluminum oxide and CH₂Cl₂/THF/MeOH; 10/5/1 as eluent.

[0068] Solvent was evaporated under reduced pressure. The rest wasdissolved in 100 ml of water. Lithium hydroxide was added to the aqueoussolution to reach pH 11. The resulted solution was extracted three timeswith 100 ml of dichloromethane. Organic layer was extracted with 20%aqueous lithium hydroxide and water and dried over anhydrous sodiumsulfate. After evaporation of the solvent, crude aminocryptand 2 wasdissolved in 200 ml of methanol. To resulted solution, 12 g of4-vinylbenzaldehyde in 40 ml of methanol was added over a 1 hour period.Reactants were refluxed in methanol for 6 hours in presence of 10 mg of4-t-butylcatechol. Methanol solution was filtered and cooled down to −5°C. 10 g of sodium borohydride was added slowly to resulted solution. Thereaction was continued under reflux for 24 hours. Methanol wasevaporated under reduced pressure and the residue was mixed with 80 mlof water; pH was brought to 1.5 with ice cold 30% methanesulfonic acid.The resulted solution was extracted three times with 150 ml of ether.Aqueous layer was brought to pH 11 with lithium hydroxide and extractedthree times with 100 ml of dichloromethane. Combined organic fractionswere extracted with water, dried over sodium sulfate and filtered.Solvent was evaporated under reduced pressure.

EXAMPLE 2

[0069] This example describes functionalizing the support substrate Z bydepositing a hydrophilic layer for binding to a derivatized cryptand.

Hydrophilic Layer Formed on Surface of Polymeric Particles Suitable forGrafting of Cryptand Monomer

[0070] 2.3 g of a dried 55% cross-linked macroporous resin (substrate isethylvinylbenzene cross-linked with 55% divinylbenzene, resinpreparation described in U.S. Pat. No. 4,224,415) was dispersed in 3.3 gof tertiary butyl alcohol (Fluka). To this slurry was added 0.37 g ofvinylbenzylacetate made in house, 1 g of vinylacetate (Aldrich) and0.092 g of Vazo 64 initiator (Dupont). The entire material was dispersedhomogeneously and then placed in an oven at 60° C. for 18 hours. Theresultant polymeric material was washed with water, acetone, water andfinally with acetone. After hydrolysis, this material is now ready forgrafting with a cryptand monomer as shown in Example 3 below.

EXAMPLE 3

[0071] This example describes binding of the derivatized cryptand ofExample 1 to the functionalized Z of Example 2.

Cryptand Monomer is Attached to Polymeric Particles Suitable for Use asa Packing

[0072] 2.35 g of a dried 55% cross-linked macroporous resin withpreformed hydrophilic layer (substrate is ethylvinylbenzene cross-linkedwith 55% divinylbenzene, resin preparation described in U.S. Pat. No.4,224,415) was dispersed in 3.4 grams of water and 0.5 g of 0.1 Mmethanesulfonic acid was added. To this slurry was added 0.5 g ofcryptand monomer and 0.2 g of azobiscyanopentanoic acid (Fluka). Theentire material was dispersed homogeneously and then placed in an ovenat 50° C. for 20 hours. The resultant polymeric material from above waswashed with acetone followed by methanol, water, and 1M potassiumhydroxide. The resin was then packed in an analytical column usingstandard methods and apparatus at 6000 psi for 15 minutes. Thispolymeric column is suitable for chromatographic separations of anionicspecies.

EXAMPLE 4 Cryptand Monomer is Attached to Polymeric Particles Which HaveNo Hydrophilic Layer Pre-Attached

[0073] In this example, the derivatized cryptand of Example 1 is boundto the macroporous resin starting material of Example 2 (without formingthe hydrophilic layer).

EXAMPLE 5 Alternate Hydrophilic Layer Formed on Surface of PolymericParticles Suitable for Grafting of Cryptand Monomer

[0074] 2.3 g of a dried 55% cross-linked macroporous resin (substrate isethylvinylbenzene cross-linked with 55% divinylbenzene, resinpreparation described in U.S. Pat. No. 4,224,415) was dispersed in 3.3 gof tertiary butylalcohol (Fluka). To this slurry was added 2.4 g ofvinylbenzylacetate (made in house), 0.092 g of Vazo 64 initiator(Dupont). The entire material was dispersed homogeneously and thenplaced in an oven at 60° C. for 18 hours. The resultant polymericmaterial was washed with water, acetone, water and finally with acetone.After hydrolysis, this material is now ready for grafting with acryptand monomer as described in Example 3 above.

EXAMPLE 6

[0075] This example shows the selectivity changes that can beaccomplished by changing the eluent cation (mobile phase).

Capacity vs. Cation Form

[0076] The conditions for these chromatograms shown in FIG. 2 are:Eluent: 70 millimolar for each case, fig. A potassium, Fig. B sodium,Fig. C lithium; flow rate, 1.0 mL/min; injection volume, 25 μL;temperature, 35 deg C.; Detector, Suppressed Conductivity, ASRS®-ULTRA4-mm, External Water mode with Anion trap column (4×35 mm); Peaks: 1.Fluoride, 2 mg/L; 2. Chloride, 3 mg/L; 3. Sulfate, 5 mg/L; 4. Nitrite,10 mg/L; 5. Bromide, 10 mg/L. The column was a cryptand column of thetype described in the examples with dimensions of 4 mm I.D.×250 mmlength. The particle size was 10 micron.

EXAMPLE 7

[0077] This example shows adjustment of capacity during an analysis byusing a higher capacity mobile phase at the beginning and then changingto a lower capacity format by either a step change or a gradient. Asshown in FIG. 3, the initial mobile phase is sodium hydroxide and then astep change is made to a lithium hydroxide mobile phase, which is a verylow capacity form. The first peaks eluting off are low valencypolyphosphates and the run ends with the elution of high valency ions.The conditions for Example 7 are as follows: Polyphosphates: CapacityGradient Prototype 3 × 150, 5 μ Eluent: 30 mM NaOH for 2 minutes, stepto 30 mM LiOH Inj. Volume: 5 μL Temperature: 35° C. Detection: SuppressConductivity ASRS-ULTRA 2-mm External water mode with ATC Sample:Polyphosphoric Acid 0.1%

[0078] The benefit of this approach is that it allows one to elute veryhigh valency ions without having to increase the mobile phase ionicconcentration. The total ionic concentration throughout the run is only30 mM, a relatively low concentration. This technique is known as“gradient capacity ion chromatography” (Lamb, et al. (Editors),“Variable Capacity Columns for Gradient Elution Anion ChromatographyBased on Macrocyclic Complexes,” Advances in Ion Chromatography, Vol. 2,Century International, Franklin, Mass., pp 215-231 (1990); Lamb andSmith, “Review: Application of Macrocyclic Ligands to IonChromatography,” J. Chromatogr. 546:73-88 (1991)).

[0079] Traditional anion exchange columns require very highconcentration mobile phases when analyzing polyvalent ions such aspolyphosphates, oligosacharrides, oligonucleotides, etc. Theconcentrations needed sometimes exceed the ability of a suppressor tosuppress. By using a cryptand-based column one can eliminate largebaseline changes often seen with steep gradients or step changes withconventional approaches.

What is claimed is:
 1. An ion exchange composition formed by reactingunsaturated carbon to carbon moieties pendant from derivatized ionbinding cryptands with a support substrate under free radical activationconditions to form a covalent bond therebetween.
 2. The method of claim1 in which said covalent bond is formed between said cryptand pendantmoiety and an unsaturated carbon to carbon moiety pendant from saidsupport substrate.
 3. The method of claim 1 in which said supportsubstrate comprises an organic polymer.
 4. The ion exchange compositionof claim 1 in which said cryptand pendant carbon to carbon moiety is ona styrenic group.
 5. The ion exchange composition of claim 1 having anion binding constant of at least about 1000 in water.
 6. The ionexchange composition of claim 1 in which the only ion binding moietybound to said support substrate is a cryptand.
 7. The cryptand ionexchange composition of claim 1 in which said support substrate is inthe form of a particulate resin bed.
 8. The cryptand ion exchangecomposition of claim 7 in which said bed comprises chromatographicseparation packed bed.
 9. The cryptand ion exchange composition of claim7 in which particles in said resin bed include at least about 100microequivalents per gram.
 10. A method for making a cryptand ionexchange composition comprising: covalently bonding unsaturated carbonto carbon moieties pendant from a derivatized ion binding cryptands withunsaturated carbon to carbon moieties pendant from a support substrateunder free radical activation conditions to form covalent bond.
 11. Themethod of claim 10 in which said cryptand pendant carbon to carbonmoiety is on a styrenic group.
 12. The method of claim 10 in which saidderivatized cryptand is the only ion binding moiety bound to saidpolymeric substrate.
 13. The method of separating anions in a liquidsample comprising flowing said liquid sample through a flowthrough bedof ion exchange composition according to any of claims 1-8 to separatesaid anions.